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  • T4 Polynucleotide Kinase (3' phosphatase minus)

    Description

    T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of double- and single-stranded DNA and RNA, as well as nucleoside 3´-monophosphates (1-5). This modified version exhibits full kinase activity with no 3´ phosphatase activity (6,7).

    Highlights

    5´ phosphorylation of DNA/RNA for subsequent ligation
    End-labeling of DNA or RNA (2)
    5´ phosphorylation of 3´ phosphorylated mononucleotides to generate a substrate (pNp) that can be added to the 3´ end of DNA or RNA by ligase activity (8)
    5´ end labeling of 3´ phosphorylated oligos (9)

    Product Source

    An E. coli strain that carries a plasmid encoding the modified T4 Polynucleotide Kinase gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T4 Polynucleotide Kinase Reaction Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [32P] in 30 minutes at 37°C (1). 

    Unit Assay Conditions: 
    1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

    Reaction Conditions

    1X T4 Polynucleotide Kinase Reaction Buffer
    Incubate at 37°C

    1X T4 Polynucleotide Kinase Reaction Buffer:
    70 mM Tris-HCl
    10 mM MgCl2
    5 mM DTT
    pH 7.6 @ 25°C

    Usage Concentration

    10,000 units/ml

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1 μM ATP
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Unit Assay Conditions

    1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

    Protocols

    1. Radioactive Labeling with T4 PNK or T4 PNK 3' phosphatase minus
    2. Non-radioactive phosphorylation with T4 PNK or T4 PNK 3' phosphatase minus

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

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    Quality Control

    Quality Assurance Statement

    • Free of exonuclease, phosphatase, endonuclease and RNase activities. Each lot is tested under 5´-end-labeling conditions to assure maximal transfer of [32P].

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Exonuclease or Phosphatase Activity (33P, Radioactivity Release):
      The product is tested in a reaction containing radioactively end labeled 5´-hydroxyl terminated d(T)8. After incubation for 30 minutes less than 1% of the product is degraded by exonuclease or phosphatase activities as determined by SDS-PAGE
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • RNase Activity (2 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
    • RNase Activity (Buffer):
      The buffer is tested in a reaction containing a RNA substrate. After incubation there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.